Combinatorial synthesis is a process for producing large numbers of compounds which can be screened for possible physiological or chemical activity. Typically, it is carried out in a series of successive stages, in each of which an existing molecule is modified chemically.
Various techniques have been devised for combinatorial synthesis.
One such technique is the so-called "split synthesis" method, in which polystyrene beads are provided as solid substrates on which the reactions take place. In the split synthesis method, the beads are divided into a number of groups of equal size. A different monomer is coupled to the beads in each group. The beads are then washed, recombined, thoroughly mixed, and again divided into a number of groups of equal size. Monomers, which may be the same as or different from the original monomers, are coupled to the beads of the new groups. By repeating the process, it is possible to produce large numbers of different compounds. The collection of compounds produced by this method is referred to as a "library."
At each stage of the synthesis, identifier molecules can be attached to the beads. The identifier molecules or "tags" attached to a particular bead uniquely represent the reaction history of the bead, and enable the compound of interest on the bead to be reproduced in quantity after it is determined, by screening for activity, to warrant further investigation.
Another technique for distinguishing compounds is "array synthesis," in which the various compounds are formed at defined locations on a surface. From a knowledge of the reagents added at each location, and the sequence in which they are added, it is possible to determine the reaction history of any compound on the surface.
Robotic systems have been devised to assist in carrying out combinatorial synthesis. For example, robots have been used to automate the synthesis of equimolar peptide mixtures by split synthesis.
On a larger scale, robotic systems have been devised for the automated production of related compounds. These systems typically utilize test tube arrays, or special multi-celled reaction blocks, and include heating or cooling devices to control the temperatures of the tubes or reaction cells, devices for moving the tubes, specialized agitation devices, devices for transferring liquids to and from the tubes or reaction cells, and provisions for maintaining an inert atmosphere.
Peptide synthesis has also been carried out by a continuous flow, solid-phase synthesis method utilizing columns packed with polystyrene-based resin. The flow columns are connected in parallel to provide multiple flow paths, or are connected in series to provide a single flow path. In both cases, complex valving is required to control the flow of various reagents and solvents through the resin-packed columns.
One of the disadvantages of the split synthesis method is that it is carried out on a microscopic scale and requires specialized and expensive techniques to read the molecular tags. The split synthesis method is advantageous in that it is capable of synthesizing very large numbers of compounds. However it is also subject to statistics, in that the splitting and combination of the beads are random, and consequently there is always a statistical probability that not all of the desired variants are present in the library.
Array synthesis is also difficult to carry out in that it requires automated apparatus or masking on a microscopic scale.
The robotic apparatus used for larger scale synthesis is mechanically complex and expensive. The continuous flow, solid-phase synthesis method, used for peptide synthesis, is not well suited for the synthesis of large numbers of related compounds, and requires complex valving to control the flow of the various reagents and other fluids. Both the robotic systems and the continuous flow synthesizers are prone to failure.
Combinatorial synthesis is described in International Patent Application WO 94/08051, published Apr. 14, 1994; and Lowe, Gordon, "Combinatorial Chemistry," Chem. Soc. Rev., 1995, pp. 309-317.
Apparatus and methods for automated synthesis are described in International Patent Application WO 91/17823, published Nov. 28, 1991; Krchnak, Viktor et al., "Multiple Continuous-flow Solid-Phase Peptide Synthesis," Int. J. Peptide Protein Res. 33, 1989, pp. 209-213; Zuckermann, Ronald N. et al., "Control of the Zymate Robot with an External Computer, Construction of a Multiple Peptide Synthesizer," J. Amer. Chem. Soc., 115, 1993, pp. 2529-2531; and Zuckermann, Ronald N. et al., "Automated Tools for the Production of Non-Natural Molecular Diversity," Innovation and Perspectives in Solid Phase Synthesis, Collected Papers, 3rd International Symposium, Roger Epton, Editor, Mayflower Worldwide Ltd., Birmingham, JK. 1994, pp. 397-402.